Nitric Oxide (NO) is a gas that has multiple signalling and effector functions in mammalian tissues. Hemoglobin scavanges NO and this limits NO's biological activity; conversely, it has recently been proposed that reversible NO binding to the beta- globin cysteine 93 is allosterically linked to oxygenation and this contributes to the control of oxygen delivery in tissues. We have investigated NO transport and metabolism by hemoglobin in human subjects in vitro and in vivo. We have developed new, highly sensitive chemical and spectrophotometric assays for S- nitrosohemoglobin (SNO-Hb) and nitrosyl (heme) hemoglobin (Hb-Fe II-NO). We find that inhalation of NO by normal subjects leads to formation of significant amounts of iron-nitrosylated hemoglobin and of nitrite, with large arteriovenous gradients, suggesting that one or both of these species could deliver NO. We find that NO inhalation, after inhibition of local NO synthesis, causes peripheral (arm) increases in blood flow in normal individuals but not in sickle cell patents. The results in normal volunteers confirm that NO can be transported as a hormone and thus has the potential to be a pharmacological agent (i.e., a drug). We believe that the lack of effect in the sickle cell patients is due to the presence of circulating hemoglobin and that this contributes to the pathophysiology of this and other chronic and acute hemolytic syndromes. In recent studies we have infused nitrite into the brachial arteries of normal humanb volunteers and have shown that this increases blood flow, suggesting that nitrite could function physiologically as a source of NO and could be used pharmacologically. We find that in vitro deoxyerythrocytes and nitrite cause aortic ring preparations to dilate, suggesting a mechanism of nitrite activation by deoxyheme proteins. We also find that nitrite inhalalation in hypoxic newborn sheep lead to decreased pulmonary artery pressures and exhalation of NO; nitrite infusions in these animals leads to decreases in mean arterial blood pressure. We are currently studying the formation and compartmentalization of nitrite in the blood, in erythrocytes in particular, and whether nitrite levels may be a marker of cadiovascular risk in humans. These studies are designed to allow us to study nitrite infusions in normal human subjects and those with a variety of ischemic (including sickle cell anemia) diseases. We have shown that the maximum production of NO from nitrite occures near the p50 of hemoglobin and is dependent on the allosteric conformation of hemoglobin. We have also developed methods to measure nitrite levels precisely in human blood and have found that most of blood nitrite is contained in the red cells; currently we are studying the uptake and possible production of nitrite (and NO)[unreadable] by an eNOS-like system in the human erythrocyte. In more recent studies we have shown that dihydroascorbic acid in red cells can catalyze the production of NO from nitrite; these results may explain our finding that blood-bank stored blood retains high nitrite levels for several weeks-a[unreadable] factor which probably contributes to the physiological effects of blood transfusions. In parallel[unreadable] studies we have examined the production of NO and nitrite in malaria-infected red cells and believe that new mechanisms exist in these cells for nitrite/NO homeostasis. All of the above studies should contribute to our understanding of the role of the human erythrocyte in modulating NO bioactivity.